The important human pathogen Bordetella pertussis is the causative agent of whooping cough, which kills hundreds of thousands of babies and children every year. Despite an extensive vaccination regimen, the prevalence of B. pertussis infection has dramatically increased throughout the industrial world in recent years, leading to epidemics in the U.S. and other countries. The reason for the reemergence is unclear but has been proposed to involve adaptation of the pathogen to populations increasingly vaccinated with an acellular vaccine containing only five proteins. Despite extensive study of the pathogen, its evolution during infection and the effects of vaccination have not been directly measured. Estimates of mutation/recombination rates based on sequencing of natural isolates with unknown relatedness are dramatically different from estimates based on in vitro study of individual genes. We recently demonstrated the ability to detect individual mutations that arise during infection and measure different mutation rates during discreet stages of bacterial infection. In this proposal, we identify sequence changes that arise in B. pertussis genomes during infection, allowing for direct measurement of the mutation and recombination rates, giving unprecedented insight into the in vivo evolution of this important human pathogen. By comparing pertussis genome evolution in immunologically nave and vaccinated animal hosts, we will determine the actual in vivo mutation/recombination rates and how they are affected by vaccination of the host. In addition to reconciling discrepancies between in vitro and population-level studies, these experiments will directly address the potential for the vaccine-driven evolution of this important pathogen.